Functional analysis of 110 phosphorylation sites on the circadian clock protein FRQ identifies clusters determining period length and temperature compensation

Abstract In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites.


Importance
Circadian rhythms, found in most eukaryotes, are based on cell-autonomous, auto-regulatory feedback loops in which negative elements feed back to depress their own expression by repressing the positive elements that drive their synthesis. In Neurospora, the WCC transcription activator drives the expression of FRQ, which complexes with FRH and CK1 to repress the DNA-binding activity of WCC by promoting phosphorylation at a group of residues of WCC. The phosphorylation status of FRQ determines the circadian period length, acting independently of effects of phosphorylation on FRQ half-life. Reflecting this dominant role of phosphorylation, FRQ is subject to substantial phosphorylation at over 100 sites in a time-of-day-specific manner. However, how this plethora of phosphoevents on FRQ controls its activity in a circadian cycle is still elusive, and prior work had shown limited effects of individual phosphosite point mutants. In this study, a series of frq mutants targeting multisite phosphorylation within domains of FRQ were generated and analyzed in order to define

Introduction
Living organisms on earth are persistently under the influence of external light/dark cycles. To anticipate and, more importantly, better utilize these environmental cues, most organisms have evolved an internal cellular oscillator, the circadian clock, that integrates daily signals, such as light, temperature, and chemicals, to metabolism (Dunlap 1996;Dunlap and Loros 2006;Wang et al. 2013;Larrondo and Canessa 2018;Diernfellner and Brunner 2020;Zhang et al. 2022). Circadian clocks regulate a wide variety of physiological and molecular events in eukaryotes and certain prokaryotes (Michael et al. 2015;Karki et al. 2020;Ding et al. 2021).
Unlike light and chemicals that only function as Zeitgebers to the core clock, the temperature can impact the core oscillator in several different ways: The period length of circadian clocks remains about the same across permissive temperatures-a phenomenon commonly called "temperature compensation" that allows the clock to make accurate time measurements while temperatures undergo large variations in nature; similar to light, both temperature pulses and steps can reset the oscillator, serving as an entrainment factor for the molecular clocks (Sweeney and Hastings 1960;Francis and Sargent 1979;Edery et al. 1994;Gooch et al. 1994;Liu et al. 1997); and finally, circadian clocks can only oscillate within a limited range of temperatures, outside which the clock will be frozen at a certain phase from which rhythmicity can be resumed if the organism is returned to permissive temperatures (Njus et al. 1977).
In Neurospora, Drosophila, and mammals, the core circadian oscillator comprises a transcription and translation-based negative feedback loop: Negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) bring about repression to their transcriptional activators, WC-1 and WC-2 in Neurospora, Clock (Clk) and Cycle (Cyc) in Drosophila, and BMAL1/Circadian Locomotor Output Cycles Kaput (CLOCK) in mammals, to terminate their own expression, thereby closing the circadian feedback loop Zhang et al. 2011). For example, in Neurospora, the White Collar Complex (WCC), a heterodimer comprised of WC-1 and WC-2, serves as the transcriptional activator for the pacemaker gene frequency (frq) by binding to one of the two DNA elements in the frq promoter: the Clock box (C-box) in the dark (Froehlich et al. 2003) or the Proximal Light-Response Element (PLRE) in the light (Froehlich et al. 2002). FRQ interacts with FRQ-Interacting RNA Helicase (FRH) and Casein kinase 1 to repress the transcription activity of WCC by promoting its phosphorylation at a group of residues (Aronson et al. 1994b;Lee et al. 2000;Cheng et al. 2005;Schafmeier et al. 2005;He et al. 2006;Hong et al. 2008;Guo et al. 2009;Shi et al. 2010;Cha et al. 2011;Hurley et al. 2013;Lauinger et al. 2014;Wang et al. 2019).
Protein phosphorylation, as the most common posttranslational modification, has been implicated in regulating protein-DNA interaction, protein-protein interaction, protein turnover, enzymatic activity, and subcellular localization, all of which have been shown to control the operation of the circadian clocks (e.g. Luo et al. 1998;Diernfellner et al. 2009;Lipton et al. 2015;Robles et al. 2017;Narasimamurthy et al. 2018;Luciano et al. 2018). In the Neurospora clock, FRQ, the core pacemaker protein, undergoes dual molecular rhythms in total abundance and phosphorylation Liu et al. 2000;Ruoff et al. 2005). The dynamic phosphorylation status of FRQ is controlled by several kinases and phosphatases including the Casein kinases 1 and 2 (CK1 and CK2), Checkpoint Kinase 2 (PRD-4), Protein kinase A (PKA), Ca/CaM-dependent kinase (CAMK-1), and protein phosphatase (PP) 1, 2A, and 4 (PP1, PP2A, and PP4) (Yang et al. , 2002Brunner and Schafmeier 2006;Pregueiro et al. 2006;Dunlap 2006;He et al. 2006;Huang et al. 2007;Cha et al. 2008;Diernfellner et al. 2019;Wang et al. 2021). Newly expressed FRQ becomes progressively phosphorylated over time and is targeted eventually for degradation through the SCF-ubiquitin ligase-recruiting protein FWD-1 (He et al. 2003). FRQ undergoes extensive phosphorylation at over 100 residues in a time-of-day-specific manner Liu et al. 2000;Tang et al. 2009) that determines its activities, controls its binding partners, and, finally, leads to its inactivation Larrondo et al. 2015). Quantitative mass spectrometry analysis reveals that phosphorylation of distinct regions of FRQ occurs at opposite phases of the clock, causing opposing effects on its activity and interacting partners over time Tang et al. 2009). In vitro kinase assays revealed that CK1 and CK2 account for a large body of FRQ phosphorylation events (Tang et al. 2009). In addition to period determination at one temperature, FRQ phosphorylation and related kinases have also been implicated in temperature compensation of the clock across physiological temperature ranges (Aronson et al. 1994a;Pregueiro et al. 2005). For example, CK2 contributes to establishing the temperature compensation of the clock via FRQ phosphorylation at certain residues (Mehra et al. 2009). In a recent study, FRQ-CK1 interaction as well as CK1-and CK2-mediated FRQ phosphorylation has been noted for regulating the period length across temperatures (Hu et al. 2021). Temperature also controls the ratio of L-FRQ to S-FRQ, derived from different start codons used in translation initiation, which is crucial for maintaining rhythmicity at a low or high temperature Diernfellner et al. 2005;Colot et al. 2005).
Mutagenetic analysis of all the plethora of phosphoresidues on FRQ becomes unavoidable and urgent in order to more fully understand their roles in controlling and fine-tuning the pace of the core oscillator. To this end, we engineered and investigated a large number of frq phosphomutants covering all the known 110 phosphosites; these were then progressively dissected into smaller clusters to discover the phosphogroups important for determining FRQ activity and thus period length. Taken together, the data show that eliminating certain phosphoclusters in the N-terminal and middle regions of FRQ mainly causes period lengthening while ablation of multisite phosphorylation at the C-terminus results in an extremely short period of 14-15 hours. Interestingly, impairing phosphorylation of a cluster of residues at the C-terminus of FRQ not only shortens the period but also leads to an overcompensated clock across a set of physiological temperatures; moreover, the elimination of certain phosphosites in the middle of FRQ leads to increased period lengths at elevated temperatures as well. Furthermore, unexpectedly, one group of phosphosites on FRQ can be epistatic to another in period determination.

frq mutant generation
To lower the cost of making a large number of frq mutants, a method described in  was modified to use yeast homologous recombination-based integration of PCR fragments (Wang et al. 2014) bearing FRQ point mutations to restrictiondigested pCB05 in place of the QuickChange II Site-directed Mutagenesis Kit (Stratagene). Four primer sets were used as flanks to facilitate homologous recombination in a yeast strain (FY834) by which point mutations of frq were introduced from PCR primers. To introduce mutations to aa 1-214 of FRQ, two PCR reactions were performed: one with a forward primer "frq segment 1F" (5′-GAACCAGAACGTAGCAGTGTG-3′) and a reverse primer "#pA R" bearing a point mutation(s) to FRQ and the other using a forward primer "#pA F" which is reverse and complementary to "#pA R" and a reverse primer "frq segment 1R" (5′-GACGATGACGACGAATCGTG-3′), and then the two PCR products were co-transformed into yeast along with pCB05 ) digested with BstXI and XhoI to create a circular construct. Similarly, to introduce mutations falling in aa 215-437 of FRQ, primers "frq segment 2F" (5′-GTGAGTTGGAGG CAACGCTC-3′) and "frq segment 2R" (5′-GTCCATATTCTC GGATGGTA-3′ were used for PCRs in combination with pCB05 digested with XhoI to NruI; "frq segment 3F" (5′-GTCGCAC TGGTAACAACACCTC-3′) and "frq segment 3R" (5′-CAGCACATGT TCAACTTCAT CAC-3′) were designed for pCB05 digested with NruI and FseI (FRQ aa 438-675), and "frq segment 4F" (5′-CACCGATCTTTCAGGAGACCCTG-3′) and "frq segment 4R" (5′-CACTCAGGTC TCAATGGTGA TG-3′) work for pCB05 digested with FseI and MluI (FRQ aa 676-989). If multiple phosphosites span two or more PCR segments mentioned above, corresponding restriction enzymes and primers encompassing the region were chosen and combined for recombination in yeast. All mutations were verified by cycle sequencing at the Dartmouth Core facility. The open reading frame of frq bearing 84 phosphomutations ( frq 84A ) from ) was custom-synthesized and purchased from Genscript, and to frq 84A , additional 26 phosphosites identified in (Tang et al. 2009) were further mutated to Ala by PCR reactions using primer pairs bearing mutations to create frq 110pA . All frq mutant constructs were targeted by homologous recombination to its native locus. Plasmids verified by cycling sequencing were linearized with AseI and SspI and PCR-purified for Neurospora transformation. Neurospora transformation was performed as previously reported (Colot et al. 2006). The recipient strain used in transforming frq mutants is Δfrq::hph; Δmus-52::hph; ras-1 bd ; C-box luc at his-3, and all frq mutants made in this study were in the ras-1 bd genetic background (Belden et al. 2007) and bear a V5H6 tag at their C-termini and frq-C-box-driven codon-optimized firefly luciferase gene at the his-3 locus (Gooch et al. 2008), except for the strains in Fig. 5, all of which bear frq-C-box-driven luciferase at the csr-1 locus rather than his-3. These strains were constructed by crossing phosphomutants from  to frq-C-box-luc at csr-1.

Lambda protein phosphatase-treatment of FRQ
V5H6-tagged FRQ was immunoprecipitated with 20 μl of V5 agarose (Sigma-Aldrich, Catalog #7345) from 2 mg of centrifugationcleared lysate, FRQ-bound V5 agarose was thoroughly washed twice using the protein extraction buffer, and all supernatant was carefully removed by pipetting. To make a total reaction volume of 52 µl, 40 μl of H 2 O, 5 μl of 10×NEBuffer for Protein MetalloPhosphatases (PMP), 5 μl of 10 mM MnCl 2 , and 2 μl of lambda protein phosphatase (NEB, Catalog #P0753S) were added to the washed FRQ-coupled V5 resin. The mixture was incubated at 30°C for 30 minutes, and then 50 µl of 5 × SDS sample buffer was added and heated at 99°C for 5 minutes ).

Phos-tag gel
To better resolve FRQ phosphorylation events, Phos-tag chemical purchased from ApexBio was added at the final concentration of 20 μM to the 6.5% SDS-PAGE Tris-Glycine gel with a ratio of 149:1 acrylamide/bisacrylamide (Wang et al. 2019).

Luciferase assay
Luciferase assays were performed as previously described (Larrondo et al. 2012). 96-well plates with each well containing 0.8 ml of the luciferase assay medium were inoculated with conidial suspension and unless otherwise specified, strains in luciferase assays were cultured at 25°C and in constant light for 16-24 hours and then transferred to the dark at the same temperature for recording light signals. Bioluminescence signals were recorded with a CCD camera every hour, data were obtained with ImageJ and a custom macro, and period lengths of the strains were manually calculated. Raw data from three replicates are shown, and time (in hours) is on the x-axis while arbitrary units of the signal intensity are on the y-axis. In Fig. 4, the strains were synchronized at 20, 25, or 30°C plus light overnight and then transferred to darkness at the same temperature used in synchronization to monitor light production by a CCD camera. Strains in Fig. 5 were entrained at 25°C for two days on a 12/12 light/dark cycle before transferring to the dark at either 20, 25, or 30°C to monitor light production by a CCD camera. Luciferase assay medium contains 1 × Vogel's salts, 0.17% arginine, 1.5% bacto-agar, 50 ng/ml biotin, and 0.1% glucose. Except for Fig. 5 (see Fig. 5 legend for controls used), WT used in the luciferase assays was 661-4a (ras-1 bd , A) that contains the frq-C-box fused to the codon-optimized firefly luciferase gene (transcriptional fusion) at the his-3 locus.

A mutagenetic strategy developed to progressively explore roles of the 110 phosphosites on FRQ
A total of 110 phosphosites on FRQ (Fig. 1a) have been identified by mass spectrometry Tang et al. 2009), but mutagenetic analyses have been conducted covering only some of these phosphosites. In this study, to screen phosphosites on FRQ impacting the pace of the circadian oscillator, we adopted a strategy successfully employed in a recent publication by which a small group of phosphoresidues from over 95 sites on WCC was identified for determining the repression of WCC and thereby the closure of the feedback loop (Wang et al. 2019). To this end, we engineered a series of frq mutants (replacing Ser/Thr with Ala) covering all the 110 phosphosites in a group manner ( Fig. 1a and 1b) and then assayed the roles of these phosphoevents in period determination by tracking bioluminescence signals in real-time.

FRQ phosphorylation is detected in frq 84pA but not in frq 110pA
Although over 100 phosphosites have been reported on FRQ, it is unknown whether they represent the entirety of the phosphoevents on the protein. To this end, we first engineered two frq mutants, frq 84pA and frq 110pA in which the 84 phosphosites ) and all the 110 phosphosites Tang et al. 2009), respectively, were mutated to Ala. The circadian clock was assayed in a strain bearing a codon-optimized firefly luciferase gene driven by the frq-C-box at the his-3 locus (Larrondo et al., 2015) in which the endogenous wild-type (WT) frq gene was replaced by the engineered frq mutants. Compared with WT, both frq 84pA and frq 110pA become arrhythmic with a high amplitude of the luciferase signal (Fig. 2a), suggesting an impaired feedback loop lacking repression of frq expression caused by these mutations. The level of FRQ in frq 84pA became extremely low but was detectable compared to that in WT (Fig. 2b). FRQ phosphorylation in frq 84pA was analyzed using a modified Phos-tag system by which single phosphorylation events on WC-1 and WC-2 could be unambiguously resolved (Wang et al. 2019). To our surprise, despite elimination of all the 84 phosphorylation sites, robust FRQ phosphorylation in frq 84pA was still detected reproducibly by the Phos-tag assay especially when compared to a lambda phosphatase-treated sample (Fig. 2c), meaning that the 84 phosphosites do not include all major phosphoevents on FRQ. Similar to frq 84pA , the level of FRQ in frq 110pA is dramatically reduced but its phosphorylation totally disappeared, reflected by the same migration pattern of FRQ bands from samples treated with or without phosphatase (Fig. 2d); these data suggest that all major phosphoevents on FRQ that occur under these growth conditions have been directly or indirectly eliminated by the 110 mutations introduced. It is worth noting that FRQ stability is known to increase in mutants disrupting phosphorylation in the N-terminal and middle parts of the protein Tang et al. 2009), so the extremely reduced FRQ abundance in frq 84pA and frq 110pA suggests an undesirable side effect caused by the large quantity of mutations that have been introduced, rather than through the elimination of phosphorylation per se.

Frq mutants identify phosphoresidues affecting period lengths
To directly examine the overall effect of FRQ phosphorylation on period length, we first made two mutants, frq 57pA and frq 27pA , together encompassing all the 84 phosphoresidues ) mutated to Ala-frq 57pA encompasses 57 phosphosites falling in amino acids (aas) 1 to 682 of FRQ were mutated to Ala altogether, and frq 27pA bears Ala mutations to the 27 phosphosites in aa 683-989 of FRQ. Consistent with the arrhythmicity observed in frq 84pA and frq 110pA (Fig. 2a), frq 57pA does not develop an oscillating clock while frq 27pA displays a robust rhythm with an extremely decreased period, 14.1 hours (Fig. 3a) (Cha et al. 2011); this suggests an additive effect contributed cooperatively by multiple phosphoevents at the C-terminus of FRQ in controlling the period length. To more specifically elucidate roles of phosphorylations in smaller regions of FRQ, four additional frq mutants derived from frq 110pA were generated, each of them containing Ala mutations to phosphosites spanning ∼200-300 amino acids (Fig. 1b). In frq 1-259pA , all phosphorylatable residues between aa 1 and 259 of FRQ were changed to Ala, while keeping the remaining aa 260-989 WT and therefore potentially phosphorylatable; in frq 260-471pA , phosphosites between 260 and 471 were changed to Ala; in frq 472-708pA phosphosites between aa 472 and 708 were changed to Ala; and in frq 709-989pA , phosphosites between aa 709 and 989 were changed to Ala. Luciferase analysis showed that frq 1-259pA and frq 472-708pA exhibit a loss of rhythmicity; frq 260-471pA has an increased period length (29.4 hours), while frq 708-989pA displays a decreased period length (14.9 hours) (Fig. 3b), consistent with the circadian phenotype of frq 27pA (Fig. 3a). frq 1-259pA bears mutations in and near to the coiled-coil domain that is required for FRQ to interact with itself and other core clock components ) as well as mutations near but not within the nuclear localization signal (NLS) (Luo et al. 1998), which would seem to explain the lost rhythmicity seen in the mutant. However, that is not the case (see below: frq 115-193pA and frq 194-220pA ). Phosphorylation surrounding the coiled coil (CC) and NLS was eliminated in frq 115-193pA and frq 194-220pA , respectively, which showed periods of 20.7 and 26 hours, respectively ( Fig. 6a and  6b), suggesting that abolishing phosphorylation within or near  1a). Strains were synchronized at 25°C in the light, and after transfer to the dark at the same temperature, bioluminescence signals were recorded by a CCD camera every hour. b) FRQ, WC-1, and WC-2 expression in wild-type (WT) and frq 84pA by Western blotting (WB). c) Phos-tag gel analysis of FRQ in WT and frq 84pA . FRQ tagged with V5H6 was immunoprecipitated (abbreviated IP'ed) with V5 resin from a constant light culture at 25°C and then treated with lambda phosphatase (labeled as Ppase) to remove phosphorylation. d) similar to (c), FRQ in frq 110pA was pulled down with V5 resin from a culture grown in constant light at 25°C, lambda phosphatase and its buffer supplied by the vendor were added to the washed resin, and the mixture was incubated at 30°C for removal of phosphorylation. In the gel for Western blot, 2.5, 5, 10, or 20 µl of immunoprecipitated/phosphatase-treated products were loaded per lane; the upper blot was performed with a regular SDS-PAGE gel, while the lower one was done using a Phos-tag gel. Red arrows point to bands of the full-length FRQ after dephosphorylation, and bands below them are S-FRQ and degradation products of FRQ, which should lack part of the N-terminus because FRQ detected here by WB against V5 is tagged with V5H6 at its C-terminus.
to these domains does not completely eliminate FRQ function, and arrhythmicity in frq 1-259pA is not entirely the result of eliminating phosphorylation within and close to CC and NLS. Because L-FRQ alone is sufficient for maintaining a clock at 25°C , the arrhythmicity of frq 1-259pA should not result from disruption of S-FRQ expression, which is also supported by the robust rhythmicity noted in frq 1-114pA , albeit with a longer period (see below).
To separately follow the impact of these phosphoevents, the 110 phosphorylation sites on FRQ were further divided into eight additional frq segments (Fig. 1b), which were mutated and analyzed by real-time luciferase assays as above. Consistent with the phenotypes of frq 1-259pA and frq 472-708pA , frq 115-259pA and frq 472-615pA are arrhythmic. frq 1-114pA , frq 260-383pA , and frq 384-471pA show increased period lengths compared to WT with period lengths of 26.7, 25.8, and 25.4 hours, respectively. frq 616-708pA and frq 709-865pA showed ∼WT period lengths. The period length of frq 866-989pA is 15 hours (Fig. 3c), mostly recapitulating the short period observed in frq 27pA (Fig. 3a) and frq 709-989pA (Fig. 3b) and indicating that phosphorylation of the C-terminal tail of FRQ contributes tremendously to period length determination. Expression of FRQ, FRH, WC-1, and WC-2 in all these eight frq mutants (Fig. 3c) is comparable to that in WT ( Supplementary Fig. 1). Except for frq 616-708pA , the other seven mutants have normal FRQ-FRH interaction ( Supplementary Fig. 1). Interaction between FRQ/FRH and WC-1/WC-2 is decreased in frq 384-471pA , frq 472-615pA , and frq 616-708pA , and it becomes undetectable in frq 115-259pA ( Supplementary Fig. 1), consistent with the lost rhythmicity seen in the strain. These data indicate that ablation of certain phosphorylations in the N-terminal and middle regions of FRQ causes period-lengthening effects; conversely, removal of phosphorylations within the FRQ C-terminus results in an extremely shortened period, suggesting an autoinhibitory role for this C-terminal domain. In agreement with the period changes of the frq phosphomutants in Fig. 3c, canonical frq alleles except for frq 1 at the N-terminus of FRQ display a lengthened period, while frq 2 (bearing the same mutation as frq 4 and frq 6 at Ala 895) shows a decreased period (Feldman 1982;Aronson et al. 1994a), suggesting that these mutations may impact phosphorylation of other residues, leading to period changes, although they are not phosphorylatable per se or conversely, neighboring phosphorylation events might modulate period lengths via impacting these nonphosphorylatable but functionally crucial residues.  866-989pA in the dark at 25°C. Strains were cultured in the race tube medium bearing luciferin at 25°C in the light overnight and transferred to darkness at the same temperature for light production recording by a CCD camera.
slightly altered across a range of physiological temperatures. Compensation is a conserved characteristic observed across diverse circadian systems. To explore whether the phosphorylation clusters on FRQ regulate the core clock at other temperatures, the eight frq phosphomutants in Fig. 3c were further examined at 20, 25, and 30°C: frq 260-383pA and frq 384-471pA show a period trend similar to that seen in WT; frq 1-114pA and frq 709-865pA display constant period lengths across temperatures even more so than WT; frq 115-259pA and frq 472-615pA remain arrhythmic, and frq 616-708pA showed a decreased period at higher temperatures, indicating this strain has an undercompensated clock ( Fig. 4 and Supplementary Fig. 2). Interestingly, frq 866-989pA bearing Ala mutations at amino acids 900, 904, 915, 917, 923, 929, 931, 950, 956, 967, and 968 of FRQ demonstrates enhanced period lengths at higher temperatures and therefore has an overcompensated clock (Fig. 4, bottom left), indicating that phosphorylation of the C-terminal tail of FRQ is involved in maintaining period lengths at enhanced temperatures. This result is consistent with a recent publication showing that mutation of three CK2 in vitrophosphorylated sites not covered in this study, S980, S981, and S982, also result in an increased period at an elevated temperature (Hu et al. 2021). Alternatively, these 11 sites are located close to the PEST-2 domain of FRQ (Gorl et al. 2001), so their phosphorylation may indirectly impact its function leading to the period adjustment. It is worth noting that the number of mutations introduced to FRQ does not always correlate with the severity of the period alteration. For example, frq 866-989pA bearing 11 mutations displays a dramatically shortened period at 25°C and an overcompensated clock across the three temperatures ( Fig. 3c  and 4), while frq 616-708pA with 12 mutations still exhibits a WT period at 25°C and an undercompensated oscillator at higher temperatures, while frq 709-865pA carrying nine mutations maintains a WT period at 20, 25, and 30°C ( Fig. 3c and 4). frq 866-989pA shows a much stronger period phenotype at the higher temperature than the frq Q2 mutant which bears Ala mutations to four phosphosites 685, 800, 915, and 929 but retains normal temperature compensation (Mehra et al. 2009), suggesting that FRQ C-terminal phosphorylations contribute collaboratively to maintaining the period length across temperatures.

Combination of few key phosphosites on FRQ is required for temperature compensation of the clock
Given that our mutational analysis of FRQ phosphosites revealed specific domains involved in temperature compensation, we investigated at a more detailed level the involvement of single, double, or triple phosphosites on FRQ in temperature compensation. A subset of the FRQ phosphosite mutants constructed in  were crossed to the C-box-luciferase reporter targeted to the csr-1 locus, and two siblings from each cross were screened at 20, 25, and 30°C (n = 3 at each temperature) (Supplementary Table 1). The negative control, ras-1 bd (clock WT), had normal temperature compensation, and the positive control, ras-1 bd , prd-3 (Mehra et al. 2009) was overcompensated as expected. Most FRQ phosphosites, when mutated, did not perturb temperature compensation, even when period length was changed (Fig. 5a 616-708pA at 30°C was calculated using the first two cycles only. Temperature in degrees is on the x-axis, and period length in hours is on the y-axis. Raw data are shown in Supplementary Fig. 2. Statistical significance for the impact of genotype and temperature on period length was determined by a two-way ANOVA analysis for rhythmic strains: frq 1-114pA (P = 0.000486), frq 260-383pA (P = 0.093818), frq 384-471pA (P = 0.034883), frq 616-708pA (P = 2.75E-06), frq 709-865pA (P = 0.002353), and frq 866-989pA (P = 5.38E-08).
period length increased as temperature increased (Fig. 5b). Compared to S538A & S540A, the additional mutation of S548 to Ala increased the period length dramatically and also caused arrhythmicity at 30°C, suggesting that this site acts synergistically with the others in this cluster. Mutation of S573A & S574A caused modest undercompensation (Fig. 5c). Statistical differences between period lengths at low vs high temperatures determined using Student's t-test (Fig. 5d) indicate that of these mutants that were examined, no single phosphosite alone is responsible for period modulation with temperature. Rather, only mutation of a combination of several key phosphosites perturbs temperature compensation, and it appears that undercompensation or overcompensation phenotypes are determined by distinct phosphosites on FRQ.

Further defining phosphosites in the arrhythmic mutants of frq
Because eliminating phosphorylation in aa 115-259 or 472-615 resulted in arrhythmicity (Fig. 3c), additional frq mutant strains bearing fewer, more select mutations were generated to these and their neighboring regions (Fig. 6a) in order to understand the roles of these phosphoevents in period manipulation. frq 1-65pA carrying nine mutations displayed a WT period length, while frq 66-114pA with eight point mutations showed a long period length similar to that in frq 1-114pA , suggesting that the effect of phosphorylations in aa 1-114 on period length is mainly caused by those in aa 66-114 (Fig. 6a). The period of frq 115-193pA was only slightly shorter than WT, while frq 194-259pA remained arrhythmic, similar to frq 115-259pA (Fig. 6a), indicating the arrhythmicity in were screened for temperature compensation defects by crossing a transcriptional frq luciferase reporter into each strain. Strains were entrained on a 12/12 light dark cycle for 2 days at 25°C, and then transferred to the dark at 20, 25, or 30°C to record luciferase oscillations. The negative control (labeled as WT) was ras-1 bd and the positive control for temperature compensation defects was the classic overcompensation mutant ras-1 bd , prd-3. a) Nearly all strains screened showed normal temperature compensation profiles, regardless of their period lengths relative to WT at each temperature. Representative examples show WT, long, and short period lengths with normal temperature compensation (see Supplementary Table 1 for all period length data). b) Two strains were overcompensated against temperature, frq S538A, S540A and frq S538A, S540A, S548A . frq S538A, S540A, S548A was arrhythmic at 30°C. c) One strain, frq S573A, S574A , was slightly undercompensated against temperature. d) Period lengths of strains depicted in a, b, and c at each temperature tested. Two siblings from each cross were screened, n = 3 at each temperature. Student's t-test was used to determine statistical significance between period length at 20°C vs 30°C (25°C vs 30°C for frq S538A, S540A, S548A ). P-value of * is ≤ 0.05, ** is ≤ 0.01, and *** is ≤ 0.001. Strains without an asterisk above indicate that the difference is not significant. Supplementary Table 1 lists period lengths for all strains tested, including those not depicted here. Supplementary Fig. 3 shows luciferase traces for strains shown in a, b, and c. Two-way ANOVA was run to test the interaction between genotype and temperature on period length for the following strains: frq S153A (P = 0.459745), frq S538A, S540A (P = 0.00057), frq S538A, S540A, S548A (P = 0.001759), frq S541A, S545A (P = 0.54789), frq S573A, S574A (P = 0.390609), and frq S900D (P = 0.891871).
frq 115-259pA is due mainly to the loss phosphosites in aa 194-259. It seems that phosphorylation may not impact FRQ dimerization, because the period length of frq 115-193pA remains ∼WT although it bears mutations close to and within the CC domain (aa 143-176) . Although frq 472-615pA is arrhythmic (Fig. 3c), frq 472-570pA shows a long period of 46.3 hours, which, to our knowledge, is the longest period seen in frq phosphomutants to date, and frq 571-615pA shows 26.4 hours (Fig. 6a). frq 616-680pA displays a long period, 26.1 hours, and frq 681-708pA is only slightly shorter (Fig. 6a). frq 616-708pA shows an intermediate period between frq 616-680pA and frq 681-708pA , which suggests an averaging effect of two neighboring phosphorylation clusters on period length. Bearing mutations near the FFC domain, frq 616-708pA has less FRH and WCC complexed with FRQ ( Supplementary Fig. 1) but it still maintains a ∼WT period (Fig. 3c), consistent with the evidence that the amount of FRH (Hurley et al. 2013) or WCC (Liu et al. 2019) in the FFC-WCC is not a determinant of the period length, even though the feedback loop relies on their presence in the complex.
To elucidate why loss of phosphorylation between aa 194 and 259 causes arrhythmicity (Fig. 6a), two additional mutants, frq 194-220pA and frq 221-259pA , were generated and assayed by luciferase analyses. frq 194-220pA has mutations to phosphosites near the NLS (aa 194-199) but is robustly rhythmic, albeit with a longer period length (Fig. 6b), suggesting that phosphorylation does not control the nuclear localization of FRQ required for a functional clock (Luo et al. 1998). This is consistent with a prior report that FRQ phosphorylation does not significantly impact its subcellular localization (Cha et al. 2011). The arrhythmicity seen in frq 221-259pA (Fig. 6b) may be caused by elimination of sites near FCD1 (Fig. 1a), a domain required for CK1 interaction and phosphorylation of the N-terminus of FRQ (Querfurth et al. 2011). frq 472-536pA and frq 537-570pA are 6 and 13 hours longer than WT, respectively, but frq 472-570pA is ∼24 hours longer (Fig. 6b), which is significantly longer than the additive lengthening of 19 hours (6 + 13 hours), suggesting that the cumulative effect of phosphorylations on period length can be stronger than the additive effect from constituent parts. frq 472-536pA contains three mutations in and close to one of the only two regions of FRQ predicted to have secondary structures (Fig. 1b). This is also a region that comprises the FCD2, so the lengthened periods of the two mutants (frq 472-536pA and frq 537-570pA ) may be due to reduced CK1 interaction, consistent with an observation that the period length is determined by FRQ-CK1 interaction (Liu et al. 2019).

Phosphomimetics at S900 could not mimic the effect of phosphorylation at the site
Phosphomimetics by amino acid substitutions like Asp (D) or Glu (E) are a widely used strategy to simulate phosphorylation by constitutively introducing a negative charge into a domain. In Neurospora, phosphomimetics have been successfully employed to study phosphorylation of the core clock components, WC-1, WC-2 and FRQ at certain sites, such as wc-1 S971D (Wang et al. 2019), wc-2 15pD (Wang et al. 2019), and frq S548D (Baker et al. 2009), revealing interesting consequences caused by constant phosphorylation at these sites. To assess whether constitutive phosphorylation at certain sites impacts FRQ activity, several key phosphosites on the protein were mutated to Asp (D) or Glu (E) to mimic the negative charge of the phosphate group. The period length of frq S915A, S917A and frq S923A is ∼2 and 1 hour longer than WT, respectively ), whereas frq S915D, S917D, S923D and frq S915E, S917E, S923E show a WT period (Fig. 8a); similarly, frq S548A becomes 4-h longer, while frq S548D maintains a WT period ), suggesting that phosphorylation at these sites of FRQ contributes to maintaining the pace of the clock. However, unexpectedly, both frq S900D and frq S900E exhibit the same period length (18.4 and 18.9 hours, respectively) (Fig. 8b) as frq S900A (∼18 hours) ), suggesting that the structure of the phosphate group of pS900 plays a more important role than the negative charge that it carries in tuning the FRQ activity. Although the phosphate group and Asp/Glu are both negatively charged, their small structural distinctions may explain the failure of frq S900D and frq S900E as phosphorylation mimics and their behavior, instead, like phosphorylation eliminators.

Discussion
FRQ has been predicted to be a largely unstructured protein comprising many disordered regions that make most of its residues exposed and accessible by kinases in the cell (Hurley et al. 2013;reviewed in Pelham et al. 2020;Marzoll et al. 2022bMarzoll et al. , 2022a, which is consistent with a large number of phosphorylatable residues identified on it. Although over 100 phosphosites on FRQ have been unambiguously documented Tang et al. 2009) and partially confirmed by a recent publication (Horta et al. 2019), and Ala mutations to some of these phosphoresidues have been shown to alter period lengths, their functions are still largely unknown due to lack of systematic mutagenesis analyses to all of them. In this study, we generated and studied a large number of frq phosphomutants covering all 110 phosphosites, and detailed mutagenetic analyses have allowed circadian roles of these site assigned to different domains of FRQ (summarized in Fig. 9). Excluding those mutations that resulted in arrhythmicity, we found that mutating phosphoresidues in the N-terminal or middle regions of FRQ only cause increased or unaltered period lengths while removal of phosphorylated residues at the C-terminus or in the middle (the cluster of S538, S540, and S548 in Fig. 5b) of FRQ results in a decreased or elevated period length, respectively, and an overcompensated circadian clock across a physiological temperature range. Interestingly, either an additive or epistatic effect on rhythmicity has been observed when combining different groups of mutations together.
How is FRQ activity tightly tuned over the course of a day? Recent publications have strongly challenged the model in which the period length is determined by the half-life of FRQ and, instead, support that time-of-day-specific phosphorylation of FRQ finely controls its activity Larrondo et al. 2015;Liu et al. 2019;Hu et al. 2021). Lacking enzymatic activity, FRQ mainly acts as a molecular platform that recruits kinases to phosphorylate its transcription activator, WCC, thereby closing the feedback loop. An intramolecular interaction between the Nand C-termini of FRQ has been demonstrated (Querfurth et al. 2011), which might be weakened or disrupted by progressive phosphorylation at multiple sites over time, leading to decreased interaction or even dissociation between FRQ and its interactors, removal of the repression on WCC, and restarting the next circadian cycle. FRQ phosphorylation can impact its activity through two different ways: Phosphorylation occurring within or close to a domain(s) can directly alter its function and interacting partners. Most phosphosites are located in the disordered regions of FRQ, and modifications at these sites can change the overall structure of FRQ in two ways: (1) by disrupting the intramolecular interaction between its N-and C-termini, which is essential for FRQ activity (Querfurth et al. 2011) or (2) by impacting the secondary structure of FRQ and thereby its interactions with its partners (e.g. . If phosphorylation at the N-terminal and middle regions of FRQ is not allowed or occurs at a slower pace, then it is plausible that the intramolecular interaction within FRQ will be sustained longer along with the capacity of FRQ in WCC repression, in consonance with the long periods seen in the frq mutants ( Fig. 3b and 3c). Phosphorylation of the FRQ C-terminal tail plays a role in slowing down the pace of the feedback loop (Fig. 3); if this molecular brake via phosphorylation is broken, FRQ loses its capacity to promote WCC phosphorylation more quickly, causing WCC to regain its transcription activity sooner. This is reflected by the short periods seen in mutants such as frq 709-989pA and frq 866-989pA (Fig. 3b and 3c). High temperatures might be able to compensate for the loss of these phosphorylations, so the shortened period gets rescued to some extent at a higher temperature (Fig. 4).
Both FRQ and its transcriptional activator WCC are subject to extensive phosphorylations in a circadian cycle, and, similarly, activities of both protein complexes are finely controlled by multiple phosphoevents Tang et al. 2009;Wang et al. 2019). For example, WCC transcription activity in the dark is completely inhibited only when a small group of sites on both WC-1 and WC-2 are simultaneously phosphorylated (Wang et al. 2019), while a large number of phosphoevents on WCC play little or no role in the core clock but only act on lowering expression of frq Phosphomimetics of residues on FRQ shows opposite effects. a) frq S900D and frq S900E display the same period length as frq S900A at 25°C. b) frq S915D, S917D, S923D and frq S915E, S9157E, S923E show a WT period length at 25°C. and clock-controlled genes (namely circadian amplitude) (Wang et al. 2019). Similarly, although FRQ is also heavily phosphorylated at numerous sites over time, to date no single phosphomutant of frq has been found to be constantly active or inactive in a circadian cycle, suggesting that FRQ activity is indeed determined by multiple phosphoevents. However, an obvious difference between phosphorylation on FRQ and WCC is that most wcc phosphomutants do not show substantially altered period lengths (Wang et al. 2019), whereas a large quantity of frq phosphomutants spanning the whole protein display period changes (Mehra et al. 2009;Tang et al. 2009;Larrondo et al. 2015). These observations agree with a model wherein complexing with FRH, FRQ serves as a platform recruiting kinases to phosphorylate and inhibit WCC, so multiple domains of FRQ participate in interactions with other proteins, including FRH, CKI, and FRQ itself via its FFD, FCD, and CC domains (Fig. 1b), respectively, as well as multiple regions for association with WCC (data not shown). Correspondingly, phosphorylations near or within these regions may directly or indirectly regulate these interactions. FRQ-dependent repression on WCC mainly targets the DNA-binding domain and its nearby regions of WCC (Wang et al. 2016(Wang et al. , 2019, which explains why mutations to phosphosites in other parts of WCC do not dramatically impact the period length. FRQ phosphorylation dynamics have been investigated by quantitative mass spectrometric analyses including stable isotope labeling by amino acids in cell culture (SILAC) ) and N 15 /N 14 isotope labeling (Tang et al. 2009). A cluster of residues surrounding the PEST-2 region (near aa 795-929) becomes hyperphosphorylated at CT8 when the level of new FRQ and thus its activity begins to increase. Eliminating phosphorylation in 709-989 (frq 709-989pA ) results in a short period (Fig. 3b), suggesting that phosphorylation in this region may be required for FRQ to repress WCC. Sites specific to the N-terminus of L-FRQ become phosphorylated at CT16, a late time point in a circadian cycle; sites in the PEST-1 domain (aa 537-558) become hyperphosphorylated later, peaking at CT12, suggesting that these phosphorylations may function in inhibiting FRQ activity. Consistent with these, frq 1-114pA and frq 537-570pA develop long periods of 26.7 and 34.8 hours, respectively (Figs. 3c and 6b). Phosphorylation of aa 211-257 peaks earlier and decreases relatively over time, suggesting that the dynamics of phosphorylation at these regions correlates with and may impact the change of FRQ activity in a circadian cycle ), supported by the arrhythmicity seen in frq 221-259pA (Fig. 6b). Due to scarcity of purified FRQ for in vitro studies and potential ionization issues of peptides bearing multisite phosphorylations in mass spectrometry, whether phosphorylation of FRQ at many sites changes in concert in a circadian cycle is still largely unknown, which restricts our understanding of the role of time-specific phosphoclusters on FRQ. Results in this work may inform understanding of mammalian and insect clocks, many facets of which are also built on timespecific multisite phosphorylation events to the key components (reviewed in Brenna and Albrecht 2020). PER/TIM in Drosophila and PERs/CRYs in mammals act as the negative elements in the negative feedback loop by inhibiting Clk/Cyc and CLOCK/BMAL1 activities, respectively, terminating their own expression and thereby closing the circadian negative feedback loop. Similar to FRQ and WCC in Neurospora, PER/TIM and PERs/CRYs also undergo extensive phosphorylation, and phosphorylation of PER/TIM and PERs/CRYs has been shown to be a critical mechanism in controlling both the fly and mammalian clocks (Chiu et al. 2008(Chiu et al. , 2011Lamia et al. 2009;Top et al. 2016;Cao et al. 2021;Cai et al. 2021;An et al. 2022). The strategy adopted here to progressively dissect scores of phosphosites on FRQ might be applicable to facilitating identification of essential phosphoevents on core clock components in other systems.
Lastly, we noted that in the case of FRQ phosphorylation at S900, an aspartic acid or glutamic acid substitution could not faithfully mimic the effect of phosphorylation (Fig. 8b); also, in a few mutants, large numbers of mutations introduced to FRQ might result in undesirable side effect(s) to the protein beyond phosphorylation elimination. These data provide a caveat to the simple interpretation of any phosphosite mutation.

Data availability
The Neurospora strains generated in this study are available upon request. Supporting material is deposited at G3 online. All data used to draw conclusions of the article have been provided within the figures and tables.
Supplemental material available at G3 online.